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Interaction of Anesthetics with Neuronal PDZ Domains

麻醉药与神经元 PDZ 结构域的相互作用

基本信息

批准号：
7850412
负责人：
Roger A Johns
金额：
$ 11.33万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-17 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7850412
关键词：
AMPA Receptors Address Affect Affinity Affinity Labels Alveolar Anesthesia procedures Anesthetics Anterior Area Attenuated Behavioral Binding Binding Sites Biological Brain Breathing C-terminal Calorimetry Chimeric Proteins Co-Immunoprecipitations Complex Data Detergents Dose Event Excitatory Postsynaptic Potentials Figs - dietary Fingers Funding GluR2 subunit AMPA receptor Halothane Hybrids In Vitro Ions Isoflurane Kinetics Knock-out Knockout Mice Label Learning Ligands Link Mediating Memory Methods Molecular Molecular Target Motor Activity Mus Mutate Mutation N-Methyl-D-Aspartate Receptors N-Methylaspartate Neuraxis Neurons Nitric Oxide Synthase Type I Peptides Persistent pain Play Potassium Channel Process Prosencephalon Protein Family Proteins Publishing Reflex action Relative (related person)Research Research Personnel Resistance Role Scaffolding Protein Signal Pathway Signal Transduction Signaling Protein Slice Spinal Cord Spinal cord posterior horn Stroke Structure Subcellular structure Surface Surface Plasmon Resonance Synapses Synaptic Transmission Titrations Work Yeasts affinity labeling central sensitization chronic pain clinically relevant dimer discs, large (Drosophila) homolog 2 protein, rat grasp in vivo neurotoxicity neurotransmission novel patch clamp postsynaptic programs protein protein interaction receptor research study response sevoflurane yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Anesthetics affect the central nervous system by altering synaptic transmission, but the mechanisms are poorly understood. PSD-95/SAP90 is one of a family of proteins recently shown to physically link synaptic signaling proteins into macromolecular signal transduction structures via PDZ domain interactions. We discovered PSD-95/SAP90 interacts with NMDA receptors and neuronal NOS in the spinal cord. Suppression of PSD-95/SAP90 expression reduced the dose required for inhalational anesthesia. Preliminary data shows clinically relevant concentrations of anesthetics dose-dependently inhibit the PDZ domain-mediated protein interaction between PSD-95 or PSD-93 and the NMDA receptor or neuronal NOS. These inhibitory effects are immediate, potent, reversible, and occur at a hydrophobic peptide-binding groove on the surface of the second PDZ domain of PSD-95. The focus of this proposal is to understand the mechanism by which inhaled anesthetics interact with PDZ domains in neuronal signaling pathways and the biological consequences of these interactions. The aims of the current proposal will: 1) Determine if the effect of inhalational anesthetics on neuronal PDZ domain-mediated protein-protein interactions observed with the NMDA receptor can be generalized to other neuronal signaling pathways using yeast 2-hybrid, co- IP, GST pulldown and plasmon resonance approaches. 2) Define the importance of inhalational anesthetic disruption of PDZ domain-containing protein interactions in the biologic state of anesthesia by investigating the effect of knockout, knockdown and direct inhibition of PSD-93/PSD-95 on MAC of inhalational anesthetics and on locomotor activity, including placing reflex, grasping reflex and righting reflex. Determine the effect of inhalational anesthetic on NMDA mediated neurophysiologic responses in the spinal cord and cortex of mice with and without knockout of PSD-95 or PSD-93. 3) Characterize the biophysical interaction of anesthetics with the PDZ domains. To do so, the effect of mutation of PDZ2 inhalational anesthetic-binding sites and other similar domains on inhalational anesthetic binding to the domains will be evaluated using plasmon resonance, affinity photolabeling, and calorimetry studies.

描述（由申请人提供）：麻醉药通过改变突触传递影响中枢神经系统，但其机制尚不清楚。PSD-95/SAP90是最近发现的通过PDZ结构域相互作用将突触信号蛋白物理连接到大分子信号转导结构的蛋白家族之一。我们发现PSD-95/SAP90在脊髓中与NMDA受体和神经元NOS相互作用。抑制PSD-95/SAP90的表达可减少吸入麻醉所需的剂量。初步数据显示，临床相关浓度的麻醉剂剂量依赖性地抑制PSD-95或PSD-93与NMDA受体或神经元NOS之间PDZ结构域介导的蛋白质相互作用。这些抑制作用是直接、有效、可逆的，发生在PSD-95第二PDZ结构域表面的疏水肽结合槽上。本提案的重点是了解吸入麻醉剂与神经元信号通路中PDZ结构域相互作用的机制以及这些相互作用的生物学后果。本研究的目的是：1)确定吸入麻醉剂对神经元PDZ结构域介导的蛋白-蛋白相互作用的影响是否可以通过酵母2-杂交、共IP、GST下拉和等离子体共振方法推广到其他神经元信号通路。2)通过研究PSD-93/PSD-95基因敲除、敲低和直接抑制对吸入麻醉药MAC和运动活动（包括放置反射、抓握反射和翻身反射）的影响，明确吸入麻醉药破坏含PDZ结构域蛋白相互作用在麻醉生物学状态中的重要性。测定吸入麻醉剂对PSD-95或PSD-93基因敲除和未敲除小鼠脊髓和皮层中NMDA介导的神经生理反应的影响。3)表征麻醉药与PDZ结构域的生物物理相互作用。为此，PDZ2吸入麻醉剂结合位点和其他类似结构域的突变对吸入麻醉剂与这些结构域结合的影响将通过等离子体共振、亲和光标记和量热研究来评估。